Uncovering the Mechanistic Role of DNA Pol Theta in UV-Damage Repair

揭示 DNA Pol Theta 在紫外线损伤修复中的机制作用

• 批准号: 10360128
• 负责人: Jamie B. Towle-Weicksel
• 金额: $ 39.16万
• 依托单位: RHODE ISLAND COLLEGE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-27 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10360128
• 关键词: Active Learning; Affect; Affinity; Authorship; Biochemical; Biophysics; Bypass; Cell Line; Cell Survival; Cells; Cellular biology; Chromosome abnormality; DNA; DNA Damage; DNA Repair; DNA biosynthesis; DNA lesion; DNA-Directed DNA Polymerase; Data; Data Analyses; Defense Mechanisms; Discrimination; Disease; Educational Status; Endogenous Factors; Ensure; Environmental Risk Factor; Enzyme Kinetics; Enzymes; Exogenous Factors; Experimental Designs; Exposure to; Fluorescence Resonance Energy Transfer; Fostering; Foundations; Functional disorder; Genes; Genome; Genome Stability; Genomic Instability; Genomics; Goals; Impairment; In Vitro; Kinetics; Knowledge; Learning; Lesion; Malignant Neoplasms; Manuscripts; Maps; Methods; Molecular Conformation; Monitor; Mutagenesis; Mutation; Normal Cell; Nucleotides; Patients; Phenotype; Play; Polymerase; Preparation; Publications; Pyrimidine Dimers; Regulation; Research; Research Personnel; Rhode Island; Role; Scientific Advances and Accomplishments; Skin; Skin Cancer; Structure; Sun Exposure; Survival Rate; System; Techniques; Testing; Time; UV Radiation Exposure; UV induced; UV induced DNA damage; Ultraviolet Rays; Variant; carcinogenesis; college; experience; experimental study; in vivo; insight; mutant; novel; overexpression; polymerization; repaired; skills; skin cancer prevention; success; therapy resistant; tumor; tumorigenesis; ultraviolet damage; ultraviolet lesions; undergraduate research experience; undergraduate student

PROJECT SUMMARY It is thought that many diseases, including cancer, begin at the DNA level. DNA is constantly being bombarded by endogenous and exogenous factors, with UV rays as one of the most powerful agents. The cell's mechanism of defense is a class of enzymes known as DNA polymerases which repair DNA damage and are regarded as guardians of the genome. Of these DNA polymerases, DNA Polymerase Theta (POLQ) has been shown to repair UV damage including the common lesions cis-syn cyclobutane–pyrimidine dimer (CPD) and a (6-4) photoproduct (PP). Despite this role, POLQ introduces mutations during this type of repair, and it is unclear if it protects the genome or contributes to genomic instability. The objective of this project is to determine the biochemical and mechanistic basis for how POLQ repairs UV-induced DNA damage through a variety of biochemical, biophysical, and cellular biology approaches. This proposal will test two hypotheses about how cancer- associated variants of POLQ in patients from sun-exposed tumors bypass CPD-damaged DNA. The knowledge gained from these experiments on the biochemical mechanisms needed to bypass UV-damage will add supporting evidence to the idea that POLQ protects again skin cancer in normal cells despite low levels of mutagenesis and that the cancer-associated variants are drivers of tumorigenesis. This is a first-of-its-kind study that explores the biochemical mechanism and cellular phenotypes of patient-derived mutants of POLQ. The long-term scientific goal of the Towle-Weicksel research group is to fully investigate how structure and function of DNA polymerases influence genomic stability and carcinogenesis. To achieve these goals, this proposal will determine the catalytic mechanism of patient-derived variants of POLQ (Aim 1) and to determine the mechanism of nucleotide selection of POLQ (Aim 2). Preliminary studies using biochemical kinetics and other cellular biology techniques suggest that one of the cancer-associated variants experiences altered DNA repair abilities and is sensitive to DNA damaging agents. These inherent differences between the variant and wild-type POLQ provide an opportunity to explore the biochemical mechanism further to expand our current understanding of how the cell copes with UV-induced damage. Undergraduate researchers from Rhode Island College will play an integral role in the success of this R15 AREA proposal. They will be involved in all aspects of the project including experimental design, data interpretation, and manuscript preparation to foster an undergraduate research experience that advances scientific discovery and cultivates scientific skills.

项目概要 人们认为，包括癌症在内的许多疾病都是从 DNA 水平开始的。 不断受到内源性和外源性因素的轰击，其中紫外线就是其中之一 最强大的防御机制是一类被称为 DNA 的酶。 聚合酶可以修复 DNA 损伤，被视为基因组的守护者。 DNA 聚合酶、DNA 聚合酶 Theta (POLQ) 已被证明可以修复紫外线损伤 包括常见病变顺式环丁烷嘧啶二聚体 (CPD) 和 (6-4) 尽管有这种作用，POLQ 在这种类型的修复过程中引入了突变，并且 目前尚不清楚它是否保护基因组或导致基因组不稳定。 该项目的目标是确定生物化学和机制基础 POLQ 如何通过各种生物化学、生物物理和 该提案将测试关于癌症如何发生的两个假设。 暴露在阳光下的肿瘤患者中 POLQ 的相关变异绕过了 CPD 损伤的 DNA。 从这些实验中获得的有关绕过所需生化机制的知识 紫外线损伤将为 POLQ 再次预防皮肤癌的观点提供支持证据 尽管突变水平较低，但正常细胞且与癌症相关的变异是驱动因素 这是一项首次研究，探讨了肿瘤发生的生化机制。 POLQ 患者衍生突变体的细胞表型。 Towle-Weicksel研究小组将全面研究DNA的结构和功能如何 聚合酶影响基因组稳定性和致癌作用。 为了实现这些目标，该提案将确定催化机制 POLQ 的患者衍生变体（目标 1）并确定核苷酸的机制 使用生化动力学和其他细胞学进行 POLQ 的选择（目标 2）。 生物学技术表明，其中一种与癌症相关的变异经历了 DNA 修复能力和对 DNA 损伤剂的敏感性之间存在这些固有差异。 变异型和野生型POLQ提供了探索生化机制的机会 进一步扩大我们目前对细胞如何应对紫外线引起的损伤的理解。 罗德岛学院的本科研究人员将在成功中发挥不可或缺的作用 他们将参与该 R15 AREA 提案的各个方面，包括 实验设计、数据解释和手稿准备，以培养本科生 促进科学发现和培养科学技能的研究经验。